{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T18:14:07Z","timestamp":1790878447296,"version":"4.1.0"},"reference-count":61,"publisher":"Wiley","issue":"12","license":[{"start":{"date-parts":[[2019,11,5]],"date-time":"2019-11-05T00:00:00Z","timestamp":1572912000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"funder":[{"DOI":"10.13039\/501100012166","name":"National Basic Research Program of China","doi-asserted-by":"publisher","award":["2016YFD0500103"],"award-info":[{"award-number":["2016YFD0500103"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100012166","name":"National Basic Research Program of China","doi-asserted-by":"publisher","award":["2017YFB0203405"],"award-info":[{"award-number":["2017YFB0203405"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["21873034"],"award-info":[{"award-number":["21873034"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["31672569"],"award-info":[{"award-number":["31672569"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["31672566"],"award-info":[{"award-number":["31672566"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003819","name":"Natural Science Foundation of Hubei Province","doi-asserted-by":"publisher","award":["2017CFA059"],"award-info":[{"award-number":["2017CFA059"]}],"id":[{"id":"10.13039\/501100003819","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100012226","name":"Fundamental Research Funds for the Central Universities","doi-asserted-by":"publisher","award":["2662016PY070"],"award-info":[{"award-number":["2662016PY070"]}],"id":[{"id":"10.13039\/501100012226","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100012226","name":"Fundamental Research Funds for the Central Universities","doi-asserted-by":"publisher","award":["2662018JC027"],"award-info":[{"award-number":["2662018JC027"]}],"id":[{"id":"10.13039\/501100012226","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100012226","name":"Fundamental Research Funds for the Central Universities","doi-asserted-by":"publisher","award":["2015RC008"],"award-info":[{"award-number":["2015RC008"]}],"id":[{"id":"10.13039\/501100012226","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["faseb.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["The FASEB Journal"],"published-print":{"date-parts":[[2019,12]]},"abstract":"<jats:p>\n                    Coronaviruses (CoVs) infect humans and multiple other animal species, causing highly prevalent and severe diseases. 3C\u2010like proteases (3CL\n                    <jats:sup>pro<\/jats:sup>\n                    s) from CoVs (also called main proteases) are essential for viral replication and are also involved in polyprotein cleavage and immune regulation, making them attractive and effective targets for the development of antiviral drugs. Herein, the 3CL\n                    <jats:sup>pro<\/jats:sup>\n                    from the porcine epidemic diarrhea virus, an enteropathogenic CoV, was used as a model to identify novel crucial residues for enzyme activity. First, we established a rapid, sensitive, and efficient luciferase\u2010based biosensor to monitor the activity of PDEV 3CL\n                    <jats:sup>pro<\/jats:sup>\n                    <jats:italic>in vivo.<\/jats:italic>\n                    Using this luciferase biosensor, along with confirming the well\u2010known catalytic residues (His41 and Cys144), we identified 4 novel proteolytically inactivated mutants of PDEV 3CL\n                    <jats:sup>pro<\/jats:sup>\n                    , which was also confirmed in mammalian cells by biochemical experiments. Our molecular dynamics (MD) simulations showed that the hydrogen bonding interactions occurring within and outside of the protease's active site and the dynamic fluctuations of the substrate, especially the van der Waals contacts, were drastically altered, a situation related to the loss of 3CL\n                    <jats:sup>pro<\/jats:sup>\n                    activity. These data suggest that changing the intermolecular dynamics in protein\u2010substrate complexes eliminates the mechanism underlying the protease activity. The discovery of novel crucial residues for enzyme activity in the binding pocket could potentially provide more druggable sites for the design of protease inhibitors. In addition, our in\u2010depth study of the dynamic substrate's envelope model using MD simulations is an approach that could augment the discovery of new inhibitors against 3CL\n                    <jats:sup>pro<\/jats:sup>\n                    in CoVs and other viral 3C proteases.\u2014Zhou, J., Fang, L., Yang, Z., Xu, S., Lv, M., Sun, Z., Chen, J., Wang, D., Gao, J., Xiao, S. Identification of novel proteolytically inactive mutations in coronavirus 3C\u2010like protease using a combined approach. FASEB J. 33, 14575\u201014587 (2019).\n                    <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"https:\/\/www.fasebj.org\">www.fasebj.org<\/jats:ext-link>\n                  <\/jats:p>","DOI":"10.1096\/fj.201901624rr","type":"journal-article","created":{"date-parts":[[2019,11,5]],"date-time":"2019-11-05T22:02:10Z","timestamp":1572991330000},"page":"14575-14587","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":50,"title":["Identification of novel proteolytically inactive mutations in coronavirus 3C\u2010like protease using a combined approach"],"prefix":"10.1096","volume":"33","author":[{"given":"Junwei","family":"Zhou","sequence":"first","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University Wuhan China"},{"name":"Key Laboratory of Preventive Veterinary Medicine in Hubei Province, Cooperative Innovation Center for Sustainable Pig Production Wuhan China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Liurong","family":"Fang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University Wuhan China"},{"name":"Key Laboratory of Preventive Veterinary Medicine in Hubei Province, Cooperative Innovation Center for Sustainable Pig Production Wuhan China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhixiang","family":"Yang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University Wuhan China"},{"name":"Key Laboratory of Preventive Veterinary Medicine in Hubei Province, Cooperative Innovation Center for Sustainable Pig Production Wuhan China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shangen","family":"Xu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University Wuhan China"},{"name":"Key Laboratory of Preventive Veterinary Medicine in Hubei Province, Cooperative Innovation Center for Sustainable Pig Production Wuhan China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mengting","family":"Lv","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University Wuhan China"},{"name":"Key Laboratory of Preventive Veterinary Medicine in Hubei Province, Cooperative Innovation Center for Sustainable Pig Production Wuhan China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zheng","family":"Sun","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University Wuhan China"},{"name":"Key Laboratory of Preventive Veterinary Medicine in Hubei Province, Cooperative Innovation Center for Sustainable Pig Production Wuhan China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiyao","family":"Chen","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University Wuhan China"},{"name":"Key Laboratory of Preventive Veterinary Medicine in Hubei Province, Cooperative Innovation Center for Sustainable Pig Production Wuhan China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dang","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University Wuhan China"},{"name":"Key Laboratory of Preventive Veterinary Medicine in Hubei Province, Cooperative Innovation Center for Sustainable Pig Production Wuhan China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jun","family":"Gao","sequence":"additional","affiliation":[{"name":"Agricultural Bioinformatics Key Laboratory of Hubei Province, College of Informatics, Huazhong Agricultural University Wuhan China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shaobo","family":"Xiao","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University Wuhan China"},{"name":"Key Laboratory of Preventive Veterinary Medicine in Hubei Province, Cooperative Innovation Center for Sustainable Pig Production Wuhan China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2019,11,5]]},"reference":[{"key":"e_1_2_8_2_1","first-page":"42","volume-title":"Coronaviridae. Andrewes' Viruses of Vertebrates","author":"Pereira H.","year":"1989"},{"key":"e_1_2_8_3_1","first-page":"1075","article-title":"Coronaviridae: the viruses and their replication","volume":"1","author":"Holmes K. V.","year":"1996","journal-title":"Virology"},{"key":"e_1_2_8_4_1","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMra032498"},{"key":"e_1_2_8_5_1","doi-asserted-by":"publisher","DOI":"10.3201\/eid1911.131172"},{"key":"e_1_2_8_6_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.01244-13"},{"key":"e_1_2_8_7_1","doi-asserted-by":"publisher","DOI":"10.1136\/vr.100.12.243"},{"key":"e_1_2_8_8_1","doi-asserted-by":"publisher","DOI":"10.3201\/eid1801.111259"},{"key":"e_1_2_8_9_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.00430-15"},{"key":"e_1_2_8_10_1","doi-asserted-by":"publisher","DOI":"10.1007\/s11262-012-0713-1"},{"key":"e_1_2_8_11_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0264-410X(99)00059-6"},{"key":"e_1_2_8_12_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrmicro2147"},{"key":"e_1_2_8_13_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.00003-17"},{"key":"e_1_2_8_14_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.02514-15"},{"key":"e_1_2_8_15_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pbio.0030324"},{"key":"e_1_2_8_16_1","doi-asserted-by":"publisher","DOI":"10.1001\/jama.1997.03540260059037"},{"key":"e_1_2_8_17_1","doi-asserted-by":"publisher","DOI":"10.1038\/nature02099"},{"key":"e_1_2_8_18_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0403596101"},{"key":"e_1_2_8_19_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi0361766"},{"key":"e_1_2_8_20_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.chembiol.2004.08.011"},{"key":"e_1_2_8_21_1","doi-asserted-by":"publisher","DOI":"10.1021\/jm0494873"},{"key":"e_1_2_8_22_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.chembiol.2004.07.013"},{"key":"e_1_2_8_23_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.chembiol.2005.01.006"},{"key":"e_1_2_8_24_1","doi-asserted-by":"publisher","DOI":"10.1021\/ja504096m"},{"key":"e_1_2_8_25_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jmedchem.7b00426"},{"key":"e_1_2_8_26_1","doi-asserted-by":"publisher","DOI":"10.1021\/ct400603p"},{"key":"e_1_2_8_27_1","doi-asserted-by":"publisher","DOI":"10.1021\/jacs.6b06454"},{"key":"e_1_2_8_28_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.str.2018.07.004"},{"key":"e_1_2_8_29_1","doi-asserted-by":"publisher","DOI":"10.1021\/acschembio.5b00647"},{"key":"e_1_2_8_30_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0066248"},{"key":"e_1_2_8_31_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.02105-13"},{"key":"e_1_2_8_32_1","doi-asserted-by":"publisher","DOI":"10.2144\/000112036"},{"key":"e_1_2_8_33_1","doi-asserted-by":"publisher","DOI":"10.2174\/1875397300802010016"},{"key":"e_1_2_8_34_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbrc.2017.05.063"},{"key":"e_1_2_8_35_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.bpj.2009.12.4272"},{"key":"e_1_2_8_36_1","doi-asserted-by":"publisher","DOI":"10.1007\/3-7643-7339-3_9"},{"key":"e_1_2_8_37_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M311744200"},{"key":"e_1_2_8_38_1","volume-title":"AMBER 2015.","author":"Case D. A.","year":"2015"},{"key":"e_1_2_8_39_1","doi-asserted-by":"publisher","DOI":"10.1002\/jcc.20290"},{"key":"e_1_2_8_40_1","doi-asserted-by":"publisher","DOI":"10.1063\/1.445869"},{"key":"e_1_2_8_41_1","doi-asserted-by":"publisher","DOI":"10.1002\/jcc.20289"},{"key":"e_1_2_8_42_1","doi-asserted-by":"publisher","DOI":"10.1016\/0021-9991(77)90098-5"},{"key":"e_1_2_8_43_1","doi-asserted-by":"publisher","DOI":"10.1063\/1.1332996"},{"key":"e_1_2_8_44_1","doi-asserted-by":"publisher","DOI":"10.1063\/1.464397"},{"key":"e_1_2_8_45_1","doi-asserted-by":"publisher","DOI":"10.1063\/1.470117"},{"key":"e_1_2_8_46_1","doi-asserted-by":"publisher","DOI":"10.1016\/0263-7855(96)00018-5"},{"key":"e_1_2_8_47_1","doi-asserted-by":"publisher","DOI":"10.1002\/prot.340110305"},{"key":"e_1_2_8_48_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0014-5793(99)01220-X"},{"key":"e_1_2_8_49_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.febslet.2009.02.003"},{"key":"e_1_2_8_50_1","doi-asserted-by":"publisher","DOI":"10.1099\/0022-1317-83-3-595"},{"key":"e_1_2_8_51_1","doi-asserted-by":"publisher","DOI":"10.1093\/emboj\/cdf327"},{"key":"e_1_2_8_52_1","doi-asserted-by":"publisher","DOI":"10.1099\/vir.0.19424-0"},{"key":"e_1_2_8_53_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gky419"},{"key":"e_1_2_8_54_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.virol.2016.04.018"},{"key":"e_1_2_8_55_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.1085658"},{"key":"e_1_2_8_56_1","doi-asserted-by":"publisher","DOI":"10.1021\/cb400100g"},{"key":"e_1_2_8_57_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1006370107"},{"key":"e_1_2_8_58_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.00377-11"},{"key":"e_1_2_8_59_1","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2015.043"},{"key":"e_1_2_8_60_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.111165798"},{"key":"e_1_2_8_61_1","volume-title":"Fields Virology","author":"Lai M.","year":"2001"},{"key":"e_1_2_8_62_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.01348-12"}],"container-title":["The FASEB Journal"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1096\/fj.201901624RR","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/full-xml\/10.1096\/fj.201901624RR","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/faseb.onlinelibrary.wiley.com\/doi\/pdf\/10.1096\/fj.201901624RR","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,28]],"date-time":"2025-10-28T21:51:23Z","timestamp":1761688283000},"score":1,"resource":{"primary":{"URL":"https:\/\/faseb.onlinelibrary.wiley.com\/doi\/10.1096\/fj.201901624RR"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,11,5]]},"references-count":61,"journal-issue":{"issue":"12","published-print":{"date-parts":[[2019,12]]}},"alternative-id":["10.1096\/fj.201901624RR"],"URL":"https:\/\/doi.org\/10.1096\/fj.201901624rr","archive":["Portico"],"relation":{},"ISSN":["0892-6638","1530-6860"],"issn-type":[{"value":"0892-6638","type":"print"},{"value":"1530-6860","type":"electronic"}],"subject":[],"published":{"date-parts":[[2019,11,5]]},"assertion":[{"value":"2019-07-01","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2019-09-23","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2019-11-05","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}]}}